PROJECT SUMMARY/ABSTRACT. The current standard of care for the X-linked bleeding disorder hemophilia is intravenous (IV) infusion of recombinant factor VIII (FVIII, for hemophilia A) or factor IX (FIX for hemophilia B). These protein products are expensive, require frequent repeated IV injections (which is painful and inconvenient), and are often targeted by antibody (?inhibitor?) responses; thereby complicating/neutralizing therapy, creating immunotoxicities, and further increasing costs. In fact, inhibitor formation, an antigen-specific CD4+ T helper cell-driven B cell response, is widely considered the most serious complication of current therapy for hemophilia. Forming an interdisciplinary research team to address this problem, our laboratories have been closely collaborating since 2007 to develop a bioengineering-based approach. We conceived and now validated the concept that oral delivery of bioencapsulated FVIII and FIX antigens produced by the chloroplasts of transgenic plants could suppress inhibitor formation in animal models of hemophilia. FVIII and FIX antigens were effectively delivered to the epithelium of the small intestine when expressed as fusions to a transmucosal carrier, and subsequently taken up by dendritic cells (DCs) in the lamina propria (LP) and Peyer's patches (PP). Tolerance was established (over a wide range, including very low antigen doses) by induction of multiple subsets of regulatory T cells (Treg) in an IL-10 dependent manner. In addition, this approach prevented and also reversed anaphylaxis against FIX. Importantly, we were able to generate chloroplast transgenic edible crop plants (lettuce), and scale up of production was successful to a commercial system that is used to cost- effectively generate GMP material. To further advance this approach, we propose the following specific aims: 1. Generate the next generation of transplastomic edible crop plants expressing FVIII antigen using cutting-edge chloroplast genomics tools and alternative transmucosal carriers. 2. Continue to define the mechanism of oral tolerance induction/immune regulation, in part through use of a model antigen. 3. Identify optimal plants for oral tolerance induction in hemophilia A mice; perform translational studies in hemophilia A dogs; and further strengthen oral tolerance by enhancing immune regulation or manipulating T cell metabolism. This work will facilitate translation of oral tolerance for hemophilia into clinical trials, define the mechanism of how plant cell-based oral tolerance is accomplished, pave the way for future combination protocols for optimal oral tolerance induction, and further advance genetic engineering of plants for biomedical applications.